Oxidative metabolism is the means by which all eukaryotic cells convert extracellular substrate (in the form of carbohydrates, lipids, and some amino acids) into adenosine 5′-triphosphate (ATP) to meet cellular energy demands. Under normal conditions, ATP production meets demand, thus pathways involved in ATP synthesis are well controlled and respond quickly to changes in energy requirements. This process of metabolizing substrate into ATP can be divided into a three stage process (as described by Jafri et al. Annu. Rev. Biomed. Eng. 2001 3:57–81). In the first stage, energy substrate is delivered across the mitochondrial inner membrane to the tricarboxylic acid (TCA or Krebs) cycle via glycolysis of carbohydrates, β-oxidation of fatty acids, and conversion of amino acids into pyruvate or TCA cycle intermediates. In the second stage, the TCA cycle in the mitochondrial matrix links glycolysis to oxidative phosphorylation (OxPhos) through decarboxylation of pyruvate to acetyl-coenzyme A (CoA) and the complete oxidation of acetyl-CoA to CO2 (see FIG. 1) In the third stage, oxidative phosphorylation (OxPhos) oxidizes reducing equivalents produced by the TCA cycle via the electron transport chain as a means of establishing a large electrochemical proton gradient across the mitochondrial inner membrane (see FIG. 2 for schematic of the OxPhos system and the interrelationship to the TCA cycle). This proton motive force is subsequently used by ATP synthase to couple the flow of protons into the mitochondrial matrix with the phosphorylation of adenosine 5′-diphosphate (ADP) to form ATP.
During ischemia or hypoxia, normal oxidative metabolism is jeopardized, with the risk of cell injury and cell death increasing with increased duration of ischemia or hypoxia. Sudden occlusion of an artery results in oxygen deprivation to the region downstream of the occlusion. This is followed by physiological and metabolic changes that begin within seconds, with the following sequence of events known to occur in a well-studied model of coronary occlusion in dogs (Kloner et al. Circulation 2001 104:2981–2989).
As taught by Kloner et al. (Circulation 2001 104:2981–2989), after about 8 seconds of decreased arterial blood flow, the O2 trapped in the tissue as oxyhemoglobin and oxymyoglobin has been consumed and energy metabolism shifts from aerobic or mitochondrial metabolism to anaerobic glycolysis. Effective contractions begin to decrease and finally stop, and the myocardium stretches, instead of shortening, with each systole. The membrane potential decreases and electrocardiogram (ECG) changes can be observed.
Kloner et al. (Circulation 2001 104:2981–2989) also teaches that the energy demands of myocytes greatly exceed the supply from anaerobic glycolysis and reserves of high-energy phosphate (HEP). Thus, tissue ATP and creatine phosphate decrease and ADP and inorganic phosphate and hydrogen ions begin to accumulate. Creatine phosphate, a major reserve source of HEP, decreases rapidly with 90% being exhausted after 30 seconds of ischemia. ATP levels decrease more slowly with approximately 20% to 25% of the ATP present at the onset of ischemia still being present late in the reversible phase of ischemia. Approximately 80% of the new HEP in zones of severe ischemia is produced by anaerobic glycolysis. Glucose-1-P from glycogenolysis serves as the substrate in anaerobic glycolysis since little glucose is present in the extracellular fluid. The process of anaerobic glycolysis generates 3 μmol HEP per μmol of glucose-1-P (Kloner et al. Circulation 2001 104:2981–2989).
After about 10 minutes of ischemia, intracellular pH decreases to 5.8–6.0, and the load of intracellular osmotically active particles, lactate, inorganic phosphate, creatine, etc, increases markedly (Kloner et al. Circulation 2001 104:2981–2989). Only a modest increase in intracellular H2O is observed, however, since relatively little H2O is available in the extracellular space of severely ischemic tissue. This edema can be viewed by transmission electron microscopy as an increase in the sarcoplasmic space.
The adenine nucleotide pool is also degraded as the ADP formed from the action of ATPases accumulates and the rephosphorylation of ADP to ATP via anaerobic glycolysis is slowed by acidosis and lactate and the diffusion of adenosine into the extracellular fluid. Various substances including bradykinin, opioids, norepinephrine, and angiotensin, are also released into the extracellular fluid during the first few minutes of ischemia. Like adenosine, these agents bind to receptors on myocytes and stimulate intracellular signaling system responses. These reactions occur quickly. For example, phosphorylase is activated only a few seconds after the onset of ischemia by the norepinephrine that is released from intramyocardial sympathetic nerve endings as a response to ischemia (Kloner et al. Circulation 2001 104:2981–2989).
Calcium is involved in, and is essential for, triggering contraction. Its balance is critical to the cell, however, as overload of Ca2+ causes hypercontraction, precipitation of Ca2+ phosphate in the mitochondria, and ultimately cell death. In the isolated perfused heart, late in the reversible phase of ischemia, intracellular ionic Ca2+ rises slightly (Kloner et al. Circulation 2001 104:2981–2989). This has been difficult to confirm, however, in vivo.
Restoration of arterial flow, also known as reperfusion, to ischemic living myocardium results in restoration of aerobic metabolism and salvage of the ischemic myocytes (Kloner et al. Circulation 2001 104:2981–2989). Upon reperfusion, the tissue develops reactive hyperemia caused by a 400% to 600% increase in blood flow. This increased blood flow reaches a peak during the first 5 minutes of reperfusion and then declines to normal control levels over the next 10 to 15 minutes. Excess O2-derived free radicals also appear during the first minute of reperfusion and peak approximately 4 to 7 minutes after the onset of reperfusion. Generalized mitochondrial and cell swelling can be observed via electron microscopy during this period. ECG changes observed during ischemia disappear after 1 to 2 minutes of arterial reperfusion and a large amount of ATP is produced via rephosphorylation of the ADP and AMP that accumulated while the tissue was ischemic. Lactate decreases to control levels and the pH of the tissue returns to normal levels approximately 0.5 to 2 minutes after reperfusion (Kloner et al. Circulation 2001 104:2981–2989).
Preconditioning (PC), a phenomenon which exists in all species examined, including humans, is a form of protection wherein a brief ischemic or hypoxic episode prevents or reduces the extent of cellular or organ damage, death and/or cellular dysfunction from subsequent prolonged ischemia. PC may also be recruited pharmacologically using agonists such as adenosine and diazoxide. PC may also occur from other events and/or agents causing cell death, damage and/or cellular dysfunction. Preconditioning occurs in various organs and tissues including, but not limited to, myocardium, skeletal muscle, smooth muscle, liver, brain and kidney.
For example, adenosine is released from cells immediately with ischemia and affects organs such as the heart as well as the vascular system through a second messenger signaling cascade triggered by binding to adenosine A1, A2a, A2b and/or A3 receptors. In the heart, adenosine affects the intrinsic conducting system (bradycardia and AV block potential arrhythmia). In myocytes it affects the Ca2+ current (negative inotropic) and has been proposed to influence the function of mitochondrial KATP channels-. It can affect the vascular system as well causing vasodilation. Adenosine causes preconditioning, potentially through activation of protein kinase C (PKC) and modulation of the mitochondrial and/or sarcolemmal KATP channel (Cohen et al. Annu Rev Physiol 2000 62:79–109), although the underlying mechanism remains controversial.